Protein concentration determination is at the heart of any study concerned with the catalytic efficiency of an enzyme. Even for highly purified enzymes the choice of first-principle methods for accurately measuring molar concentrations is restricted to a few techniques (amino acid, total nitrogen, and absorbance measurement (Pace et al., 1995), titration of oxidized sulfur (Guermant et al., 2000). For enzymes in crude solution the options are even smaller and techniques are much more elaborate (e.g., active-site titrations involving the stoichiometric release of a reporter group, enyme-linked immunosorbent assay (ELISA), densitometry after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)). Catalytic rate assays while highly specific for an enzyme and often quantitative in nature presuppose validation with purified enzyme which in turn requires first-principle methods for accurate mass quantitation.
The determination of the concentration of a specific protein among other proteins in crude solution, such as a fermenter broth, is a formidable challenge. Even more demanding is the task of verifying the presence of a specific protein and the quantitation of this protein in a cell or tissue extract without knowing the properties of the protein and ever having seen it before.
Most methods for estimating protein concentration are built on general properties of proteins, e.g., the chemistry and light absorbance of aromatic side chains and the peptide bond, and the binding affinity for chromophores. More specific techniques, e.g. immunoassay and active site titration, require some prior knowledge of the targeted protein. All such methods, however, suffer from interferences, as the extensive literature on protein assays documents, and none of the methods takes advantage of that one unique feature that differentiates non-identical proteins, the amino acid sequence. On that level there is no interference possible.
The use of isotopically labeled biopolymers to investigate cellular processes is not new. For example, Chowdhury et al. used mass spectrometry and isotopically labeled analogs to investigate the molecular weight of truncated mature collagenase, and Stocklin et al. have investigated human insulin concentration in serum samples that had been extracted and purified. Neither one discuss the use of crude solutions to determine biopolymer concentration without prior isolation of the biopolymer.
The present invention makes use of the subunit sequence as a unique tag of a biopolymer (e.g., the amino acid sequence of a specific protein), that can be exploited for determining the concentration in crude solutions.